We have recently demonstrated in the vascular smooth muscle cell (VSMC) that Ca2+-signaling regulates both Boyden assay chemotaxis, and the prerequisite calcium/calmodulin-dependent protein kinase II (CaMK II) activation, in response to PDGF. Since significant heterogeneity in the rate of chemotaxis is evident among proliferating VSMCs (i.e., only 5-10% chemotaxis at 4 hrs), we sought to understand how intracellular calcium (Cai) signaling is regulated among these cells and coordinated with other intracellular signaling pathways including kinase trafficking, and the resulting morphological polarization and reorganization of the cytoskeleton during chemotaxis. We have developed a novel microscope-adapted Boyden chamber which allows simultaneous measurement of locomotion and Cai in individual cells during chemotaxis (through filters with 8 _m pores). In addition, the ability to perfuse this chamber enables periodic intervention with experimental agents throughout the assay. Experiments with this chamber have demonstrated that significant differences in the spatio-temporal patterns of Cai exist between migrating and non- migrating cells. There are two distinct, sequential cellular Cai signaling phases required for VSMC chemotaxis: (1) an initial phase (time frame of minutes), with a relatively synchronous, transient increase in Cai in most cells, associated with CaMK II activation, and (2) a delayed phase (time frame of hours), with asynchronous and heterogeneous increases in Cai among cells which precede (and whose timing apparently determines) VSMC chemotaxis. During the initial phase, focal adhesion kinase (pp125FAK), and phosphatidylinositol 3-kinase (PI3K)(which we found requisite for chemotaxis), translocate to the activated cell membrane "patch" over filter pores, and this polarization may be important in coordinating cytoskeletal remodeling during chemotaxis. The actin-cytoskeleton normally undergoes dynamic reorganization during chemotaxis. This occurs as a series of transitional phases from organized actin stress fibers in the stationary cell to various forms of membrane ruffling in the migrating cell, accompanied by the formation of a cortical actin shell during actual PDGF-gradient chemotaxis, with the eventual reformation of stress fibers upon completion of migration, beyond the PDGF-gradient. The delayed-phase Cai- increase in individual cells coincides with abrupt pore transmigration (a process prevented in cells with Cai buffered by an intracellular Ca2+-chelator) and could provide the signal necessary for the generation of sufficient force by contraction of the cortical actin shell to overcome the cellular resistance to passage through the pore. Preliminary evidence, consistent with CaMK II co-association with actin stress fibers, suggests a plausible mechanism whereby local or global Cai activation, via CaMK II, could signal and coordinate dynamic actin reorgan-ization. Thus, the close coordination of Cai and specific kinase activation and perhaps trafficking appears critical in the orchestration of cytoskeletal dynamics responsible for PDGF-directed VSMC chemotaxis.